Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Crytochrome b₅-lipid interactions. Includes bibliographical references (p. 301-310). Also available online through Digital Dissertations.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Gregory Raner; submitted to the Dept. of Chemistry and Biochemistry. Title from PDF t.p. (viewed Jun. 7, 2010). Includes bibliographical references (p. 58-59).

The structure and function relationships involved in the binding of quinones to the terminal oxidases of Escherichia coli, cytochromes bd and bo3, were investigated using redox potentiometery, site-directed mutagenesis and magnetic resonance techniques. A stable semiquinone was identified as an intermediate of quinol oxidation by cytochrome bd in appropriately poised samples of both the membrane-bound and purified enzyme reconstituted with excess ubi- and menaquinone analogues. The effects of the inhibitors HOQNO and aurachin D on semiquinone formation were assessed. The potentiometric behaviour of the semiquinone stabilised by membrane-bound and purified cytochrome bo3, reconstituted with excess quinone analogues was characterised. The effects of two cytochrome bo3 subunit II mutations and the inhibitor tridecylstigmatellin were studied. The data presented is consistent with the presence of one quinone binding site. The hyperfine splittings present in the ESR spectrum of the cytochrome bo3 semiquinone were resolved by ENDOR spectroscopy. The resultant electronic structure of the bound semiquinone and the nature of the hydrogen bonding to the protein is described. ESEEM spectroscopy was used to identify a nitrogen nucleus hydrogen bonded to the semiquinone. A model of this quinone binding site and a possible location is presented.

No description available.

The cytochrome o and cytochrome d oxidase complexes provide twin termini for the branched respiratory chain of aerobically grown Escherichia coli. Combined use of mutant strains, modulated growth conditions and high resolution analytical techniques enabled cytochromes to be resolved, identified and partially characterized. The cytochrome complement of everted membrane vesicles and detergent extracts fractionated by liquid chromatography is more complex than previously recognised. Multiple type-b cytochromes were resolved by potentiometry and by high resolution spectrophotometry in membrane vesicles from mutant strains lacking the cytochrome d oxidase complex and grown under conditions minimising respiratory chain diversity. Cytochrome o was identified with Em = +235 mV (vs. NHE) as were low potential cytochromes associated with dehydrogenases. Spectrally distinct components of the cytochrome d complex yielded Em values of +125 mV (cytochrome 6595) and +187 mV (cytochrome d). The latter displayed atypical redox behaviour with extreme hysteresis during potentiometric titrations. Several cytochromes b₅₅₆ displaying single, symmetrical redox α-bands at 77 K were resolved from detergent extracts of vesicles. Mutant strains identified one with Mr = 52500 (gel filtration) and Em = +20 mV as the sdhC gene product, a component of succinate dehydrogenase. DL-lactate induced another while a hydroperoxidase, Mr = 386000 (gel filtration) with twin Em values of -2mV and -121 mV and a split Soret absorption band at 77 K (λ[symbol omitted]max= 426.0 nm + 434.0 nm) was produced under limited oxygen tension. The Triton-solubilized and purified cytochrome 0 complex exhibited Mr = 516000 (gel filtration) with five component peptides of Mr= 55000, 32000, 31000, 21000 and 16000 (SDS-PAGE). It displayed mid-point potentials of -58 mV, +127 mV and +260 mV and three a-absorption maxima at 77 K : 554.5 nm, 557.0 nm and 563.5 nm. These components were reduced equivalently during poised-potential low temperature spectrophotometric analyses. Carbon monoxide binding changed the complex's redox α-absorption spectrum minimally but shifted the high potential Em to approximately +420 mV. Quinone analogues inhibited both reduction and reoxidation of the complex. Cytochrome o complex prepared from cloned sources contained a significantly greater proportion of the component with mid range electrochemical potential absorbing at 554.0 nm. These results are discussed in relation to possible structures of the complex, its respiratory interactions and the identity of cytochrome o itself. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Escherichia coli can produce several respiratory chains which transfer electrons to oxygen, nitrate or other acceptors. Cytochromes are amongst the electron carriers of these chains, and can be studied by a number of spectroscopic techniques. Of particular interest is the formate-nitrate reductase respiratory pathway which is composed of two complexes, formate dehydrogenase and nitrate reductase each with a cytochrome associated. The cytochromes of E. coli after growth under a variety of conditions have been studied by spectrophotometry redox titration and other spectroscopic methods. These have shown that, contrary to some previous reports, there are at least six cytochromes produced irrespective of the culture conditions used. This emphasizes the need for simplified systems and two of these have been investigated. By fractionation of cytochromes prior to spectroscopic analysis a cytochrome b of redox potential OmV was identified in cells grown 556 aerobically. In addition the association of two cytochromes of potential +20mV and +120mV with nitrate reductase was demonstrated. Secondly the formate-nitrate reductase pathway has been investigated by spectroscopic studies of chi mutants which are defective in this activity. Three phenotypes of cytochrome production were observed. Mutants at loci associated with the production of the cofactor for both nitrate reductase and formate dehydrogenase were shown to produce the same cytochromes as the wild-type. Mutants mapping at the chlC locus and defective in nitrate reductase but not formate dehydrogenase were found to lack only the two cytochromes associated with nitrate reductase. They had high leyels of a cytochrome of redox potential -100mV which was shown to be associated with formate dehydrogenase. A third class of pleiotropic regulatory mutants was identified which was not related with a specific genotype. These produced none of the anaerobic respiratory pathways but overproduced the aerobic respiratory pathway leading to cytochrome d. The second aerobic respiratory pathway leading to cytochrome o was most evident in double mutants lacking both of the cytochromes associated with nitrate reductase and that associated with formate dehydrogenase. On the basis of these results a model for the arrangement of the cytochromes in the respiratory chains of E. coli is proposed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The degradation of cytochromes P-450 by fluroxene (2,2,2-trifluoroethyl vinyl ether) has been investigated. Fluroxene is shown to specifically degrade cytochromes P-450 in vivo and in vitro without affecting the levels of the other microsomal enzymes, cytochrome ~S a.nd NADPH-cytochrome~ reductase. Fluroxene appears to degrade the haem moiety of cytochromes P-450 but does not affect the level of the apoprotein. The degradation of cyto-chromes P-450 by fluroxene is accompanied by a loss of E-nitroanisole 0-demethylase and biphenyl 4-hydroxylase activities and a decrease in the extent of aniline binding is observed. By using cytochromes P-450 dependent reactions which are catalysed by specific type P-450 cytochromes,~.~· the hydroxylation of benzpyrene, the N-demethylation of ethyl-morphine and the binding of ethyl isocyanide, it is established that only cytochrome P-450 is degraded by fluroxene in vivo following phenobarbital induction of animals, and both cytochrome P-450 and cytochrome P-448 following methylcholanthrene induction. The same type P-450 cytochromes are shown to be degraded by fluroxene in vitro in phenobarbital and methylcholanthrene induced microsomes. This was established from studies of the kinetics of the fluroxene mediated degradation of cyto-chromes P-450. In addition, the K values for the flurox-m ene mediated degradation of cytochromes P-450 differ with iii the different inducing agents and indicate the involve-ment of two different type P-450 cytochromes in the degradation reaction in methylcholanthrene induced micro-somes. Metabolic activation of cytochromes P-450 by the cyto-chromes P-450 drug metabolising pathway appears to be essential for the fluroxene mediated degradation of cyto-chromes P-450. Since none of the known or proposed metabolites of fluroxene can mimic the degradation of cytochromes P-450 by fluroxene, a reactive species is proposed to be involved. By varying the experimental conditions, and with the use of inhibitors of cytochromes P-450, the likely sequence of events in the fluroxene mediated degradation of cytochromes P-450 is shown to be as follows: fluroxene is metabolised by cytochrome P-450 to a transient reactive intermediate which has the ability to degrade the haem moiety of cytochrome P-450 and cyto-chrome P-448. By comparing the ability of various analogues of fluroxene to degrade cytochromes P-450, it is established that the formation of the proposed reactive intermediate is dependent on the presence of the vinyl moiety of the molecule. Initial studies indicate that the reactive species may take the form of an epoxide.

Thèse de doctorat : Génétique humaine : Paris Est : 2008. / Titre provenant de l'écran-titre.

No description available.

No description available.